This invention relates to a method of calculating the rate of increase in the products of enzymatic reactions. More particularly, the invention relates to a method which, in the utilization of enzymatic reactions to quantitate substances as in enzyme immunoassays, is capable of providing continuous calibration curves for overall concentration range while enabling highly precise quantification of a substance of interest in the low-concentration range.
Methods of quantitating substances utilizing enzymatic reactions are currently utilized in many biochemical and immunological tests. For example, the general practice in enzyme immunoassays comprises mixing a specimen such as human serum with an immobilized antibody against an analyte and an enzyme-labelled antibody against the analyte to form an immune complex, causing the enzyme in the complex to react with a substrate, measuring the concentrations of the resulting product of the enzymatic reaction to determine the rate of its increase and comparing the measured values with a calibration curve preliminarily constructed by performing similar procedures on a standard sample containing known concentrations of the analyte.
In practice, the reaction product is not directly measured but signals correlated to its concentration such as the intensity of fluorescence and the absorbance of light are measured.
Problems with the methods of quantitating substances utilizing enzymatic reactions are that when an enzyme is allowed to react with its substrate for a prolonged time, the concentration of the enzyme's substrate decreases and as the concentration of the reaction product increases, the rate of its increase decreases, and that during the reaction the enzyme has the potential to be inactivated over time under the influence of the components in the reaction mixture. Decreases of the similar kinds may also occur depending upon the detection system employed.
Under the circumstances, in most methods of quantitating substances utilizing the rate of increase in the enzymatic reaction products, it is common practice to measure the product of enzymatic reaction within limits (a concentration range) where the measurement is not highly sensitive to the effects of the concentrations of the substrate and the reaction product while ensuring that the time of measurement is restricted within limits where the enzyme will not be inactivated by the components of the reaction mixture. If these conditions are conformed, the concentration of the reaction product vs the point of time of measurement can be approximated by a linear function and the slope of this linear function is the rate of increase in the reaction product, or commonly termed the "rate of enzymatic reaction".
However, the rate of increase in the reaction product which is calculated using the values obtained by measurements within said limits of concentration range and time is sensitive to slight variations of measurement value and conditions and this has been a problem in biochemical and immunonological tests which require good reproducibility and high precision in measurement.
If such restricted conditions are not observed but the range of measurement is expanded to those concentrations of the reaction product at which the concentration of the substrate or the reaction product is influential on the enzyme's activity, the relationship between the time of measurement and the concentration of the reaction product becomes curvilinear and cannot appropriately be approximated by a linear function. If the range of measurement time is increased with a view to reducing the variations in the calculated values of rate, more of the measured values will exceed the upper limit of measurement with the detector from the stage where the enzyme's activity is comparatively low. Therefore, if measurements are conducted at discontinuous points of time and if the obtained values of measurement are approximated by a linear function, with its slope being equated as the rate of increase in the enzymatic reaction product, the rate of increase calculated when the number of effective measurements changes will change in a non-continuous manner and a plot of the rate against enzyme's activity may potentially lose continuity or smoothness (the continuity of differential values).
This phenomenon could be avoided if the measured values obtained by measurement at continuous points of time were approximated by a linear function; however, in the areas of biochemical and immunological tests where large volumes of samples have to be quantitated, there is a need to process many samples and if the detector is occupied for a long period by procedures for a single sample, the processing speed decreases to eventually cause the problem of a failure to meet the aforementioned requirement.
Another problem with the quantitation of substances utilizing enzymes is that since a plurality of measurement systems involve variations in operating parameters such as the amount of a sample to be dispensed, the amount of the reaction mixture, the reaction temperature and the state of agitation of the reaction mixture, it is necessary to perform quantification in collation against the calibration curve obtained with an identical measurement system. This enables consistent measurements over a broad range of concentrations; however, if the measurement is conducted at discontinuous points of time with the result being approximated by a linear function as described above, the continuity and smoothness of the calibration curve is lost rather than maintained and if, in order to solve this problem, measurements are made at continuous points of time, the rapidity in measurement which is required in areas such as biochemical and immunological tests cannot be insured.